132 Egypt. J. Chem. Vol. 64, No 3. pp.

1433 - 1439 (2021)

Egyptian Journal of Chemistry

http://ejchem.journals.ekb.eg/

Ultrasound-Assisted Extraction of Damsin and Neoambrosin from

Ambrosia maritima: Optimization Using Response Surface Methodology

S.I.A. Abdel-dayema*, M. N. A. Khalil a,b, E. H. Abdelrahman a, H. M. El-Gohary a and A. Yehiac,d

a
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo 11562, Egypt.
b
Pharmacognosy department, faculty of pharmacy, Heliopolis University for Sustainable Development.
c
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo 11562, Egypt.
d
Chemistry Department, School of Pharmacy and Pharmaceutical Industries, Badr University in Cairo (BUC),
Badr City 11829, Cairo, Egypt.

Abstract
Damsin and neoambrosin are two important anticancer phytopharmaceuticals of Ambrosia maritima (Damsissa).
Despite their importance, no report has yet discussed their extraction from plants on lab or industrial scales.
Consequently, the present article aims to develop an ultrasonic-assisted extraction (UAE) model for their
extraction on the lab scale. Box-Behnken model was applied to investigate the effects of ethanol strength (X 1: 40-
100%), drug solvent ratio (DSR, X2: 1:20-1:60), and time (X3: 30-90 min) on the yield of damsin and
neoambrosin in damsissa extract. Surprisingly, only ethanol strength was the significant factor in damsin yield
with an optimum value of 55%. Time and DSR played no significant roles in damsin content. No statistically
significant model could be deduced for neoambrosin. The model is a preliminary step to elucidate factors
affecting the UAE of sesquiterpene lactones from plants and a preamble for its large scale green extraction.

Keywords: Damsin; Neoambrosin; Ultrasonic-assisted extraction; response Surface method; sesquiterpene lactones;
Ambrosia.

1. Introduction banks and Nile delta. It is traditionally used as

Natural products are still an important
repertoire for lead pharmaceuticals especially
anticancer drugs. Sixty percent of current
anticancer are derived from natural products
[1]. Sesquiterpene lactones (STL) are a large
class of compounds distributed in several plant
families. They act as potent anti-cancer, anti-
inflammatory, antimicrobial, and molluscicidal
[2]. Their anticancer activity is mediated
through different targets, however, inhibition
of NF- κβ was the main target [3-5]. STL act
by inhibiting the activation of NF-κβ, binding
of NF-κβ to DNA, interacting with DNA-NF-
κβ complex [4, 6-8]. Efficacy of STL paved
the way for testing them in clinical trials or to
synthesize new drugs based on their nucleus
[3, 5].
Ambrosia maritima, locally known as
damsissa, is a weed growing on Nile river
_________________________________________________________________________________________________
*Corresponding author e-mail: shymaa.aly@pharma.cu.edu.eg.
EJCHEM: Received date 01 November 2020; revised date 02 December 2020; accepted date 06 December 2020
DOI: 10.21608/EJCHEM.2020.47736.2989
©2019 National Information and Documentation Center (NIDOC)
diuretic, antidiabetic, anti-inflammatory, and
molluscicidal [9]. Damsin and neoambrosin
are two major STL found in genus Ambrosia.
They had potent anticancer activity against
many cancer cell lines and multi-drug resistant
cells [9-13]. Similar to other STL, damsin and
neoambrosin interact with NF-κβ by
methylation of cysteine-38 and Cys-120 and
rendering it unable to bind to DNA,
consequently, halting the inflammation
process, promote apoptosis of cancer cells [14,
15]. Moreover, they can interact with the
DNA-NF-κβ complex [14]. Recently, Damsin
and neoambrosin showed significant cytotoxic
activity against multidrug-resistant cell lines
by silencing several targets, c-Src kinase,
STAT3, Akt and ABC transporters; therefore
they are suggested as remedies for refractory
tumors [9] Both compounds showed a potent
antirheumatoid activity [16].
1434 S.I.A.Abdel-dqyem et.al.
_____________________________________________________________________________________________________________
Large scale extraction of STL is imperative
to secure enough supply. Chemical synthesis
of damsin required eighteen reactions and was
found intricate because of the many chiral
centres of the molecule [17-20].
Phytochemical isolation would be a cheaper
and reliable alternative to chemical synthesis.
Compared to conventional extraction
techniques, ultrasound-assisted extraction is
better in terms of saving time, energy, solvent
volume, CO2 emission [21, 22]. Although
cavitation was known as the main mechanism
of action of UAE, several other mechanisms
are involved, e.g., fragmentation, erosion
capillarity, detexturation and sonoporation
[21]. Several factors affect the yield of the
process, e.g., ultrasonic power, time,
temperature, etc. [21, 22]. Response surface
method (RSM) is a statistical method utilized
to evaluate the effect of different variables on
one or more responses of variables. This can
be achieved from a lower number of
experiments and in a shorter time compared to
other optimization methods [23-25].
The current work is the first report about the
extraction of damsin and neoambrosin from
damsissa. Ultrasound-assisted extraction was
utilized and response surface model was employed
to optimize the extraction condition. Hopefully, the
present work would be a step for scaling up the
extraction of STLs from Damsissa as well as other
plants rich in STL.
2. Experimental
2.1. Plant material & chemicals:
Ambrosia maritima aerial parts were collected
from the Botanical Garden of Faculty of Pharmacy,
Cairo University. Plant material of A. maritima was
authenticated by Prof. Dr. Wafaa M. Amer, Botany
Department, Faculty of Science, Cairo University.
A voucher specimen was prepared and deposited in
Cairo University herbarium (CAI). Another voucher
specimen (13.06.2018) was deposited in the
herbarium, Pharmacognsoy Department, Faculty of
Pharmacy, Cairo University. Samples were
powdered after being dried in shade.
Ethanol, HPLC grade was purchased from Thermo
Scientific, Germany. Deionized water prepared in
the institute. Damsin and neoambrosin authentic
Table 1: Real and coded values of UAE independent variables
Alcohol %, X1
Drug solvent ratio (DSR, X2)
Extraction time (X3).
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Egypt. J. Chem. 64, No.3 (2021)
materials were previously isolated and authenticated
[16, 26].
2.2. Model construction
Box-Behnken design was constructed for the
optimization of ultrasound-assisted extraction of
sesquiterpene lactones from A. maritima. Three
parameters were selected for optimization of the
extraction process, namely, Ethanol ratio (Ethanol
%, X1), Drug solvent ratio DSR (X2), and
extraction time (X3). Three levels for each
independent variable were selected as in table 1
The design consisted of 15 runs; however, 14
were used in analysis after the omission of an
outlier. The model proposed for each response Y
was
Y = β0 + β1X1 + β2X2 + β3X3+ β12 X1X2 + β13
X1X3+ β23 X2X3 + β11 X12+ β22 X22+ β33 X32
(equation 1)
Y is the response, i.e., damsin or neoambrosin
content (μg/g dry powder). X1, X2, X3 are the
independent variables, namely, ethanol percentage,
DSR, and time, respectively. β0 is the intercept. β1,
β1, β1, are the linear coefficients. β12, β13, β23,
are the interaction coefficients; β11, β22, β33, are
the quadratic coefficient terms.
Box-Behnken design was set up using JMP
software Statistical DiscoveryTM from SAS
(Germany). The established model was validated by
Regression value (R2), p-value (p<0.05), and lack
of fit testing. Significance of the tested independent
variables and interactions were determined form
Pareto-graph, ANOVA testing.
2.3. Extraction process:
Different weights of damsissa powder
(0.33–1.00 g) powder were extracted in 20 ml
solvent (40-100% ethanol) in an ultrasonic
bath for different durations (30-90 min)
according to individual experiments (Table 1).
Extraction was performed in Elmasonic S, 0.8
l with an ultrasonic frequency of 37 kHz (Elma
Schmidbauer GmbH, Germany) at room
temperature. Samples were extracted in 50 ml
falcon tubes, then centrifuged, aliquots of 2 ml
were separated and filtered through a
membrane filter (0.45 mm). Volumes of 100
µl were directly injected in HPLC.
-1 0 1
40 70 100
20 40 60
30 60 90
 ULTRASOUND-ASSISTED EXTRACTION OF DAMSIN AND NEOAMBROSIN FROM……
_____________________________________________________________________________________________
2.4. HPLC analysis
Damsin and neoambrosin as well as the
different extracts were analysed as previously
performed but with slight modifications [16].
HPLC Agilent 1200 infinity instrument was
equipped with an autosampler and DAD
detector. The mobile phase system consisted
of solvent A & B, water containing 0.1%
formic acid and acetonitrile, respectively. The
separation was in gradient mode on
LiChrospher 100 RP-C18 column, preceded by
RP-C18 guard column, and started by using
solvent B at 45% for 2 min, then, it was
gradually increased to attain 58% at 19
minutes then increased to 100% at 21 minutes.
Afterward, the column was washed using 100
% MeOH for 2 min. Quantification was
determined at wavelength 240 nm. Standard
curves for damsin and neoambrosin were set
up in a concentration range of 3.125-100, 1.65-
50mg/ml, respectively.
3. Results and Discussion
Few reports had studied the UAE of
sesquiterpene lactones from plants. One report
evaluated the extraction of alantolactone and
isoalantolactone [27]. In the other report, UAE
was done as a preparatory step before
percolation to extract artemisinin [28]. Neither
of the two reports had utilized the RSM to
mathematically model the extraction process.
A recent study utilized RSM to optimize the
extraction of cyanopicrin [29]. The biological
significance of damsin and neoambrosin
necessitated the search for a green isolation
technology as a preliminary step before large
scale production. HPLC analysis showed that
damsin and neoambrosin were major
components of the ethanolic extract of
damsissa (Figure 1).
Fig. 1: HPLC chromatogram of Damsissa extract. Inserts
show the chemical structure of damsin and neoambrosin.
DMS, damsin; NMS, neoambrosin.
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Egypt. J. Chem. 64, No.3 (2021)
1435
3.1. Effect of extraction variables on damsin
content.
Damsin was quantified from 47 to 723
μg/g DW. The highest content was obtained
when extraction was performed using 70%
ethanol for 90 min and DSR of 1:60. The
lowest yield was obtained when the powder
was extracted with 100% ethanol for 60 min
and at DSR of 1:20 (Table 2). UAE of damsin
can be modelled, its model had a satisfactory
R2 of regression at p<0.05 and there was no
lack of fit (Table 3). The prediction model for
Damsin (Eq.2) showed a higher positive
coefficient for ethanol percent factor (X1)
compared to other factors main effects.
ANOVA testing of individual variables
revealed that only ethanol strength was the
most significant parameter, it affected damsin
content at first order and quadratic terms
(Table 3). Other factors like DSR and
extraction time have a negative impact on
damsin extraction process. However, their
effects were not significant. Besides,
interaction between factors was also trivial by
comparison. Except for ethanol strength, no
other parameters had a significant effect on the
UAE of damsin. The response surface model
showed that decreasing the alcohol strength
positively affected the damsin content to the
optimum value of 55%. Variation in DSR has
no effect (Figure2).
Considering only the significant variables, the
predictive equation which can be utilized to
optimize the UAE of damsin from damsissa
was introduced (Eq.3).
Damsin (mg/g) = 129.22 + 22.22X1 – 5.55X2 –
4.15 X3 + 0.06X1X2 - 0.02X1X3 + 0.03X2X3 -
0.22X12 + 0.04 X22 + 0.05 X32 ,
R2 = 0.97, p value < 0.008 (Equation 2)
Damsin (mg/g) = -85.49 + 25.75X1 - 0.24X12,
R2 = 0.89, p value < 0.0001 (Equation 3)
To further validate the model, the extraction
procedure was repeated in triplicate using
ethanol strength of 55%, as deduced from the
response surface curve (Figure 2). However,
extraction time and DSR were kept to lower
levels due to their insignificance, 30 min, 1:20,
respectively. The model predicted extraction
of 587.25 μg/g damsin. Experimental result
showed close values (610.74 ± 34.4 μg/g)
which verify the validity of the model.
Although STL (SLC) is not polar, higher
solubility in EtOH/water mixtures could be
attributed to several factors. Water tends to
increase tissue swelling; hence, a larger
surface area of plant material is exposed to
solvent. Moreover, bubble formation helps in
the diffusion of hydrophobic parts of the
1436
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molecule in the hydrophilic aqueous solvents
due to the formation of gas-liquid interfaces
[30, 31]. Higher yields of alantolactone were
achieved with 70% ethanol [27]. Similarly,
60% isopropanol was the best solvent to
extract rebaudioside A from Stevia and at a
short time (18 min) [32]. Sonication time,
DSR had no significant effect in the UAE of
gallic acid, punicalin, and punicalagin from
pomegranate peels [33].
S.I.A.Abdel-dqyem et.al.
was attained when ethanol 100% was utilized
for 90 min at DSR 1:40 (Table 2). Although
this tremendous difference can be directly
related to ethanol strength, ANOVA testing of
individual variables of the polynomial model
shows no significance (Table 3). This is
probably due to the lower content of
neoambrosin compared to damsin in the
studied plant. Therefore, there is no significant
difference in the extraction content between
ethanol percent levels. The positive impact of
ethanol content on neoambrosin yield can be
deduced from its response surface model
(Figure 3). The figure also shows small
curvature of the quadratic term nonsignificant
effect. DSR ratio did not affect neoambrosin
yield (Figure 3).

Fig.2: Response surface plot for the effect of Alcohol

%/DSR on damsin content.

3.2. Effect of extraction variables on

neoambrosin content

Neoambrosin was extracted at variable

quantities ranging from 24 to 193μg/g. Its Fig. 3: Response surface plot for the effect of Alcohol
maximal level was attained when powder %/DSR on neoambrosin content
extracted with 40% ethanol for 90 min at DSR
of 1:40. The minimal yield of Neoambrosin

Table 2 Box-Behnken design with coded and real values as well as the observed responses in
different extracts
Alcohol Percent Drug solvent ratio, Damsin Neoambrosin
Run Time, X3 (min)
(A%), X1 (%) (DSR) (1-X2) (μg/g powder)* (μg/g powder)
1 1 (100) 0 (40) 1 (90) 104.44 24.246
2 0 (70) -1 (20) -1 (30) 432.53 63.019
3 0 (70) -1 (20) 1 (90) 539.75 110.94
4 1 (100) 0 (40) -1 (30) 101.24 18.595
5 0 (70) 1 (60) -1 (30) 555.57 112.34
6 -1 (40) 0 (40) 1 (90) 623.84 193.27
7 0 (70) 0 (40) 0 (60) 506.38 113.48
8 1 (100) -1 (20) 0 (60) 47.884 19.723
9 -1 (40) 0 (40) -1 (30) 565.77 155.63
10 -1 (40) 1 (60) 0 (60) 525.44 148.23
11 0 (70) 0 (40) 0 (60) 538.87 121.68
12 -1 (40) -1 (20) 0 (60) 524.47 141.53
13 0 (70) 0 (40) 0 (60) 474.59 90.563
14 0 (70) 1 (60) 1 (90) 723.32 135.55

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Table 3: Estimated regression coefficient and ANOVA of the fitted second-order polynomial models of the
investigated parameters
Damsin Neoambrosin
source p-value <0.05
Model 0.008 0.018
Lack of fit 0.139 0.333
A% (X1) 0.032 0.749
DSR (X2) 0.603 0.413
Time (X3) 0.505 0.696
X1X2 0.470 0.987
X1X3 0.675 0.463
X2X3 0.645 0.565
X12 0.004 0.229
2
X2 0.708 0.490
X32 0.297 0.562

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